Exosome fusion liver targeting liposome drug delivery system as well as preparation method and application thereof

Abstract

The invention relates to an exosome fusion liver targeting liposome drug delivery system as well as a preparation method and application thereof, and belongs to the field of high polymer material science and pharmaceutical preparations. The invention discloses an exosome-fused liver-targeted liposome drug delivery system, which combines high-concentration PEG8000 and Nycodenz in a breakthrough manner, synergistically improves the fusion efficiency of exosome and liposome, and forms bionic vesicles with uniform size and high drug loading capacity. The exosome not only can exert the anti-inflammatory and anti-oxidation characteristics, but also plays a role in resisting a severe gastrointestinal environment and crossing a biological barrier. In addition, liposome is modified by cholic acid derivatives through EDC / NHS mediated amidation reaction, precise targeting of the liver is achieved, and the liposome is fully accumulated in the liver. The effects synergistically realize the treatment of type II diabetes and non-alcoholic steatohepatitis.

Description

Technical Field

[0001] The present invention relates to an exosome-fused liver-targeted liposome drug delivery system, a preparation method thereof, and an application thereof, belonging to the fields of polymer materials science and pharmaceutical preparations. Background Art

[0002] Type II diabetes is a chronic disease caused by insufficient insulin secretion or reduced efficiency. In addition, the accumulation of inflammatory lipids and cytokines in diabetic patients can cause liver fibrosis and apoptosis, leading to non-alcoholic steatohepatitis (NASH). The NLRP3 inflammasome has been verified as a key factor promoting NASH, mainly existing in innate immune cells. NLRP3 plays an important role in synthesizing mature interleukin-1β (IL1β) and can initiate metabolic inflammation. In addition, it not only promotes the transformation of hepatic macrophages into the pro-inflammatory M1 phenotype, but also induces the release of additional NLRP3 and inflammatory factors, resulting in a harmful cycle. It has been proven that inhibiting excessive NLRP3 can reduce liver inflammation. However, there is still no specific drug treatment for obesity-induced type II diabetes and NASH.

[0003] Berberine is an isoquinoline alkaloid with antioxidant, immunomodulatory, and anti-fibrotic properties. Berberine can effectively control the postprandial blood glucose of early type II diabetes patients. Berberine can reduce lipid accumulation, improve mitochondrial function, relieve oxidative stress, etc. by injection, showing the prospect of being a potential therapeutic agent for NASH. However, its low oral bioavailability limits its application. To achieve the therapeutic effect, berberine needs to be administered at a high dose (mouse: 100-250 mg / kg / day, human: 900-1500 mg / day), but it will cause potential side effects, further limiting its clinical application.

[0004] Exosomes are spherical structures composed of a phospholipid bilayer, and their main lipid components include cholesterol, saturated fatty acids, and sphingomyelin. They can transport molecules such as proteins, nucleic acids, and lipids between cells, are often regarded as natural carriers of signaling molecules, and can encapsulate drugs. However, the retention characteristics of exosomes in the intestine and low drug-loading capacity limit their therapeutic effects. Liposomes, as effective drug delivery systems, have a high drug-loading capacity. Therefore, the combination of exosomes and liposomes can be considered in drug delivery.

[0005] However, the existing exosome-liposome fusion technologies generally face problems such as low fusion efficiency, poor product stability, and insufficient drug-loading capacity, making it difficult to meet the needs of precise treatment. For example, the chemical method often uses polyethylene glycol (PEG) mediation, and 45% - 60% of PEG is commonly used 4000 / PEG 6000Incubation has limited drug loading capacity. Moreover, the larger the relative molecular mass and the higher the volume fraction, the greater the toxicity to cells, which may cause cell deformation or even rupture, and ultimately the fusion is not uniform and complete (Journal of Hubei Normal University (Natural Science), 2014, 34(1)). Currently, there are many methods for purifying and extracting exosomes, including ultracentrifugation and density gradient centrifugation. The density gradient centrifugation method is cumbersome and time-consuming, while the ultracentrifugation method takes a long time and has a low yield. Currently, low-concentration Nycodenz can be used for gradient centrifugation in the purification process of other cells. For example, Li et al. used 20% - 34% Nycodenz to purify rat cardiac mitochondria (Journal of Zunyi Medical University, 2016, 39(5): 525 - 528), and Li et al. used 8% - 8.2% Nycodenz to isolate hepatic stellate cells (Progress in Modern Biomedicine, 2014, 14(16): 3033 - 3037). However, there are few techniques for separating plant exosomes using Nycodenz.

[0006] Meanwhile, under this technical background, the existing exosome-liposome fusion drug delivery system also has deficiencies: when using exosomes alone to carry small molecule drugs or using liposome delivery systems alone, the drug delivery efficiency and therapeutic effect of the fusion system formed by combining the two with traditional techniques in the existing technology are limited. For example, in the study of hyaluronic acid-modified liposome-exosome hybrid carrier (HL@Exo) for treating acute photo-damaged skin by Xiao et al. (International Journal of Biological Macromolecules, 2025, 306: 141606), the SOD level and MDA content in the HL@Exo treatment group did not increase much compared with those in the HL and Exo treatment groups; the chlorphosphate-nintedanib hybrid exosome-liposome composite preparation (CLD / NIN@LIEV) prepared by Ji et al. (ACS Nano 2024, 18: 21091 - 21111) showed limited performance in macrophage phenotype regulation. The CD163 / CD86 ratio in the CLD / NIN@LIEV treatment group was not much different from that in the group with CLD loaded alone, and the improvement was not significant. Although both have the ability to promote pro-inflammatory to anti-inflammatory phenotype conversion, the pro-inflammatory to anti-inflammatory ability of the final experimental group was slightly inferior. In addition, resveratrol liposomes and exosomes were fused to form hybrid nanoparticles. In the determination of hydrogen peroxide, FRAP, and ROS scavenging ability, Exo@Lip-Res, although retaining the antioxidant ability of Lip-Res, the fusion nanoparticle ability was lower than that of the Lip-Res group. Summary of the Invention

[0007] The object of the present invention is to provide an exosome-fused liver-targeted liposome drug delivery system, a preparation method thereof, and an application thereof in the treatment of non-alcoholic fatty liver disease and type II diabetes.

[0008] In order to achieve the above object of the invention, the technical solution adopted by the present invention is: an exosome-fused liver-targeted liposome drug delivery system, and its preparation process is: fusing exosomes with liver-targeted liposomes modified with bile acid derivatives to prepare empty nanoparticles, and encapsulating drugs in the empty nanoparticles to prepare liver-targeted exosome-loaded nanoparticles, and the liver-targeted exosome-loaded nanoparticles are the exosome-fused liver-targeted liposome drug delivery system.

[0009] In a preferred embodiment, the exosomes are derived from one of the following plants, including ginger, kaempferia galanga, yam, pineapple, etc.

[0010] Further, the bile acid derivatives include ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, etc.

[0011] Further, the drug is one of the drugs that can treat liver diseases, including berberine hydrochloride, morin, semaglutide, etc.

[0012] The present invention also provides a preparation method of an exosome-fused liver-targeted liposome drug delivery system, including the following steps:

[0013] (1) Preparation of liver-targeting ligand:

[0014] Put the bile acid derivative and the reactant in water, add DSPE-PEG 2000 -NH2, react at 35-39 °C for 20-24 h, and purify to obtain the liver-targeting ligand;

[0015] (2) Extraction of exosomes:

[0016] Extract ginger juice, and obtain ginger exosomes through low-temperature high-speed centrifugation and sucrose density gradient centrifugation;

[0017] (3) Preparation of liver-targeted liposomes modified with bile acid derivatives:

[0018] Dissolve the lipid mixture composed of an emulsifier, the liver-targeting ligand obtained in step (1), and cholesterol in an organic reagent, and prepare liver-targeted liposomes modified with bile acid derivatives by extrusion;

[0019] (4) Preparation of empty nanoparticle solution:

[0020] Mix the exosomes obtained in step (2) with the liver-targeted liposomes modified with bile acid derivatives obtained in step (3) and PEG 8000 Mix, co-incubate and purify by density gradient centrifugation to obtain a mixture, where PEG 8000The concentration is 60 ± 3% w / v; the mixture is mixed with 75 - 85% w / v Nycodenz to form a Nycodenz solution containing the first concentration of the mixture. The Nycodenz solution of the first concentration, the Nycodenz solution of the second concentration, and PBS form a gradient solution. After ultracentrifugation, the top separation layer is separated to obtain purified empty nanoparticles; the Nycodenz concentration gradient decreases in the range of 80% to 15% (preferably 40% to 15%, referring to the difference between the first concentration and the second concentration); the first concentration is lower than 75 - 85% w / v; the second concentration is lower than the first concentration.

[0021] (5) Preparation of ligand-modified drug-loaded nanoparticles:

[0022] Take the drug solution and the empty nanoparticle solution in equal volume and mix them to obtain liver-targeted exosome drug-loaded nanoparticles; the concentration ratio of the empty nanoparticle solution to the drug solution is 1:1 - 3.

[0023] The surface of ginger exosomes is rich in polysaccharides and low-immunogenic proteins, which are more likely to form a hydrophilic and charge-neutral hydration layer, reducing the binding to mucin glycoproteins and decreasing the probability of being trapped by mucus. The membrane flexibility of ginger exosomes is also higher than that of animal exosomes, while the membrane hardness of other plant exosomes (such as grapefruit exosomes) is relatively higher, resulting in a decrease in the penetration efficiency.

[0024] In the present invention, for the fusion of ginger exosomes and bile acid liposomes, the direct incubation method is adopted, but such methods may have problems such as increased size, uneven size distribution, and low fusion efficiency. The present invention adds a high concentration of PEG 8000 , which can achieve the purpose of purifying exosomes with high efficiency, enabling the complete exosomes to fuse with liposomes. Through multiple experimental explorations on substances, finally 80% Nycodenz (w / v) is adopted. In addition, density gradient centrifugation is used to purify the fusion product (GR-Exos) to ensure the uniformity of nanoparticles. This targeted improvement makes the fusion of exosomes and liposomes uniform. In this article, Nycodenz is provided by Axis-Shield Company in Norway and is a density gradient separation solution.

[0025] The acylation reaction of bile acid derivatives (such as ursodeoxycholic acid) with DSPE-PEG 2000 needs to precisely control the molar ratio (1:1 - 1.5), reaction time (24 hours), and temperature (37°C). Otherwise, incomplete modification or the generation of by-products will reduce the liver-targeting efficiency and cause the accumulation of drugs in non-target tissues. If the feeding order is incorrect, the main reaction is blocked: after the amino group of DSPE-PEG 2000 -NH2 is modified by EDC, it cannot be coupled with the active ester of UDCA, the generation of the main product (DSPE-PEG 2000 -UDCA) decreases, and at the same time, by-products accumulate.

[0026] The key step parameters in the preparation method include:

[0027] Exosome extraction: A three - centrifugation procedure (1000 - 10000g, 10 - 40 minutes) combined with ultra - high - speed centrifugation (200,000g, 2 hours), and the sucrose gradient needs to be precisely layered (8% - 60%).

[0028] Fusion and purification: Use 80% Nycodenz (w / v) and separate the empty - loaded nanoparticles (GR - Exos) by centrifugation with a 40% Nycodenz gradient (200,000g, 2 hours).

[0029] Drug - loading optimization: The GR - Exos solution and the berberine solution need to be mixed in a ratio of 1:1 - 3. The ultrasonic parameters are 55 - 70kHz and 15 - 30 minutes to ensure efficient drug encapsulation without damaging the nanostructure.

[0030] Preferably, the reactants are 1 - (3 - dimethylaminopropyl) - 3 - ethylcarbodiimide hydrochloride (EDC·HCl), N - hydroxysuccinimide (NHS); in step (1), the molar ratio of the reactants EDC·HCl or NHS, the bile acid derivative, and DSPE - PEG 2000 -NH2 is 1 - 1.5:1:1 - 1.5.

[0031] Further, step (2) is specifically: Extract ginger juice, centrifuge at 800 - 1200g for 5 - 10min, 2500 - 3500g for 15 - 20min, 9000 - 11000g for 20 - 40min at 0 - 4°C; ultra - centrifuge at 180,000 - 200,000g for 1 - 2h, resuspend, and transfer to sucrose solutions with different gradient concentrations for centrifugation. The gradient concentrations of sucrose are 8%, 15%, 30%, 45%, and 60% respectively; then centrifuge, collect the bands between the 8% / 30% layer and the 30% / 45% layer, wash, centrifuge, and resuspend to obtain ginger exosomes.

[0032] In step (3), the emulsifier, liver - targeting ligand, and cholesterol are mixed in a mass ratio of 1 - 5:1 - 2:2.

[0033] In step (4), the volume ratio of exosomes, liver - targeting liposomes modified with bile acid derivatives, and PEG 8000 is 1 - 2:1:1 - 2.

[0034] In step (5), the drug is encapsulated in the empty - loaded nanoparticles by ultrasound, and the ultrasonic parameters are 55 - 70kHz and 15 - 30min.

[0035] Another object of the present invention is to disclose the application of the exosome-fused liver-targeted liposome drug delivery system or the preparation method thereof in the preparation of drugs for treating non-alcoholic steatohepatitis and type II diabetes.

[0036] The present invention fuses exosomes with liposomes to form a biomimetic vesicle, which not only retains the membrane proteins of exosomes but also improves the drug loading capacity of this carrier. Liposomes modified with bile acid derivatives have liver targeting properties and can target exosomes to the liver. Drugs such as berberine hydrochloride can treat non-alcoholic steatohepatitis, but its oral bioavailability is low and it does not have liver targeting properties. The biomimetic vesicles prepared by the present invention can encapsulate such drugs, overcome gastrointestinal barriers and achieve liver targeting. At the same time, the drug and the vesicle complement each other and are used in combination to achieve long-term drug circulation, and have potential application prospects in the treatment of non-alcoholic steatohepatitis and type II diabetes.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] (1) The exosome drug delivery system designed by the present invention uses the prepared exosomes as an oral drug carrier, and has the advantages of being endogenous, biocompatible and multifunctional. Exosomes have good drug loading capacity, unique biomolecule protection, intestinal mucus penetration and tissue bioavailability, etc., and have great advantages in maintaining the stability of oral drugs and the permeability of the intestinal mucus layer.

[0039] (2) Liposomes modified with bile acid derivatives by EDC / NHS-mediated amidation reaction are taken up by endocytosis mediated by the apical sodium-dependent bile acid transporter (ASBT) and transported intracellularly by ileal bile acid binding protein (IBABP). Subsequently, it enters the enterohepatic circulation and finally specifically guides the drug to the liver, achieving precise targeting of the liver. The application of liver targeting technology can promote the accumulation of drugs in the liver and fully accumulate, thereby playing the effect of precise treatment, improving the bioavailability of oral administration and achieving long-term drug circulation.

[0040] (3) The biomimetic vesicles formed by fusing liposomes and exosomes in the present invention not only retain the membrane proteins of exosomes and improve their drug encapsulation ability. GR-Exos has the ability to specifically target NLRP3 inflammatory vesicles in liver macrophages, and uses its anti-inflammatory and antioxidant properties to inhibit NLRP3 expression. At the same time, berberine can play a synergistic role, and at a lower drug dosage, enhance the inhibition of inflammatory factors and improve NASH and type II diabetes.

[0041] (4) The present invention uses a high concentration of PEG in combination 8000Cooperate with Nycodenz to improve the fusion efficiency of liposomes and exosomes, and use centrifugation to purify to finally obtain biomimetic vesicles with uniform size and high drug loading. The method for obtaining the vesicles is simple, the materials have low oral toxicity and good biocompatibility. Description of the Drawings

[0042] Figure 1 1H NMR spectrum of DSPE-PEG 2000 -UDCA (400 MHz, DMSO-d6); 1

[0043] Figure 2 Size distribution and morphology diagrams of nanoparticles RAL, G-Exos, GR-Exos and GR-Exos@B in Example 3;

[0044] Figure 3 Fusion characterization diagrams of G-Exos and GR-Exos in Example 4(2), (A) Detection of G-Exos size by nanoparticle tracking analysis (NTA); (B) Evaluation of the integrity of GR-Exos by FRET spectroscopy;

[0045] Figure 4 In vitro release diagrams of RAL@B, G-Exo@B and GR-Exo@B in Example 4(3), (A) In a solution of pH 6.8; (B) In a solution of pH 1.2;

[0046] Figure 5 Cell viability schematic diagrams of Caco-2 and HT-29 cell toxicity tests in Example 4(4), (A) HT-29; (B) Caco-2;

[0047] Figure 6 Schematic diagram of mucus penetration of drug-loaded nanoparticles in Example 4(5);

[0048] Figure 7 Schematic diagram of the body weight change trend of diabetic mice taking samples such as GRExos@B, G-Exos@B and RAL@B for a long time in Example 5(1);

[0049] Figure 8 Schematic diagram of serum inflammatory factor levels 60 days after administration in Example 5(1), (A) LDL, (B) TG, (C) HDL, (D) AST, (E) ALP, (F) ALT;

[0050] Figure 9 Liver H&E staining, Masson staining and Oil Red O staining diagrams in Example 5(1);

[0051] Figure 10For the Western blot analysis of genes such as COA and CY2EBP-1 in the liver in Example 5(1), g. Evaluate the expression of COA and CY2EBP-1 in the liver by Western blot analysis; 1: HFD; 2: NM; 3: RAL@B; 4: G-Exos@B; and 5: GRExos@B; h. Expression of SCD, SREBP-1 and CD95; numbered as above; i. Liver AMPK, p-AMPK, Nrf2, p-NRf2, HO-1 and NOQ1, numbered as above;

[0052] Figure 11 Schematic diagram of TNF-α, IL-6, IL-4, IL-1β and IL-10 levels in each administration group in Example 5(1);

[0053] Figure 12 Schematic diagram of glucose tolerance test and insulin resistance index after 60 days in Example 5(2), (A) Test insulin tolerance after intraperitoneal injection (i.p.) of insulin into fasting mice after 60-day treatment; (B) Insulin resistance index of HFD-fed mice treated with G-Exos@B, RAL@B and GR-Exos@B; (C) Fasting and 2-h postprandial blood glucose of mice after 60-day treatment;

[0054] Figure 13 Levels of C peptide in mice of each administration group after 60-day treatment in Example 5(2); Purple: NM, Blue: HFD, Yellow: G-Exos@B, Green: RAL@B, Red: GR-Exos@B;

[0055] Figure 14 Visualization of insulin immunostaining images of islets in mice of each administration group after 60-day administration in Example 5(2), Scale bar: 30.0 μm;

[0056] Figure 15 Western blot analysis of the expression of PEPCK, PGC-1α and GLUT4 in the liver in Example 5(2), 1: NM; 2: HFD; 3: RAL@B; 4: G-Exos@B; and 5: GR-Exos@B;

[0057] Figure 16 Determination of the M1 / M2 type ratio of macrophage markers in BMDM cells by flow cytometry in Example 5(3);

[0058] Figure 17 Evaluation of NLRP3 expression in BMDM cells by Western blot in Example 5(3); 1: BBR; 2: M1; 3: M2; 4: RAL; 5: G-Exos; 6: GR-Exos, and 7: GR-Exos@B. And statistical analysis of the protein expression level of NLRP3;

[0059] Figure 18 For the hematoxylin and eosin (H&E) staining of the main organ sections of the mice in each dosing group of Example 5(4), scale bar: 40.0 μm. Detailed implementation mode

[0060] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0061] An exosome-fused liver-targeted liposome drug delivery system of the present invention is prepared by the following process: fusing exosomes with liver-targeted liposomes modified with bile acid derivatives to prepare empty nanoparticles, encapsulating drugs in the empty nanoparticles to prepare liver-targeted exosome-loaded nanoparticles, and the liver-targeted exosome-loaded nanoparticles are the exosome-fused liver-targeted liposome drug delivery system.

[0062] DSPE-PEG (polyethylene glycol-distearoyl phosphatidylethanolamine) and bile acid derivatives are reacted through amidation to obtain a ligand-modified polymer DSPE-PEG-UDCA (which is a liver-targeting ligand). At the same time, materials containing exosomes are centrifuged multiple times with an ultra-high-speed refrigerated centrifuge to obtain exosomes. Subsequently, the polymer, emulsifier, and cholesterol are dissolved in an organic solvent and then extruded with an extrusion instrument to obtain liposomes. The liposomes and exosomes are mixed to prepare ligand-modified empty nanoparticles, and the empty nanoparticles and drugs are mixed by ultrasonic means to obtain the exosome-fused liver-targeted liposome drug delivery system.

[0063] Further, the bile acid derivatives include ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, etc.

[0064] Further, the exosomes are derived from one of the following plants, including ginger, kaempferia galanga, Chinese yam, pineapple, etc.

[0065] Further, the liver-targeting ligand is connected by reacting polyethylene glycol-distearoyl phosphatidylethanolamine (DSPE-PEG 2000 ) with bile acid derivatives through amidation. The molar ratio of DSPE-PEG 2000 to bile acid derivatives is 1 - 1.5:1.

[0066] Further, for the extraction of the exosomes, first remove macromolecular substances in the exosome material with a low-temperature high-speed centrifuge, then take the supernatant and centrifuge it with an ultra-high-speed refrigerated centrifuge to remove small-molecular substances, obtain an exosome precipitate, resuspend it with PBS, and transfer it to sucrose gradient centrifugation at different concentrations, and collect the bands between different concentrations to obtain the exosome solution.

[0067] Furthermore, for the preparation of the liver-targeting liposomes modified with bile acid derivatives, a lipid mixture composed of an emulsifier, a liver-targeting ligand, and cholesterol in a ratio of 1-5:1-2:2 is dissolved in an organic solvent, incubated, and evaporated. After splitting the dried lipid film, the lipid film is hydrated, rotary evaporated at room temperature, and finally extruded using a liposome extruder to obtain the liposomes.

[0068] Furthermore, for the preparation of empty nanoparticles by fusing the exosomes with the liver-targeting liposomes modified with bile acid derivatives, the exosomes, the liver-targeting liposomes modified with bile acid derivatives, and a carrier are dissolved in a solution in a ratio of 1-2:1:1-2, incubated at a certain temperature for a fixed time, and purified GR-Exos are obtained by density gradient centrifugation.

[0069] Furthermore, for the exosome-fused liver-targeting liposome drug delivery system, the empty nanoparticles are mixed with a drug, and the resulting nanoparticle solution is obtained by ultrasonic treatment using a cell ultrasonic disruptor.

[0070] The preparation method of the exosome-fused liver-targeting liposome drug delivery system of the present invention includes the following steps:

[0071] (1) Preparation of the liver-targeting ligand:

[0072] The bile acid derivative and the reactant are placed in water, DSPE-PEG 2000 -NH2 is added, and the reaction is stirred at 37 °C. After the reaction is completed, it is precipitated with an organic solvent, centrifuged, and the lower-layer precipitate is taken, and vacuum dried to obtain the liver-targeting ligand.

[0073] (2) Extraction of exosomes:

[0074] The macromolecular substances in the material are removed by centrifugation using a low-temperature high-speed centrifuge, and then the supernatant is centrifuged using an ultra-high-speed refrigerated centrifuge to remove the small-molecular substances, obtaining an exosome precipitate. The precipitate is resuspended with phosphate buffer (PBS solution) and transferred to sucrose gradient centrifugation at different concentrations. The bands between different concentrations are collected, washed with PBS, centrifuged after balancing, and finally the precipitate is resuspended and stored at -80 °C for later use. The obtained exosome solution is denoted as G-Exos.

[0075] (3) Preparation of liposomes:

[0076] A lipid mixture composed of an emulsifier, the ligand solution obtained in step (1), and cholesterol is dissolved in an organic reagent. After water bath evaporation, the dried lipid film is split by adding an organic reagent. Then the lipid film is hydrated and sequentially extruded through a polycarbonate porous membrane using a liposome extruder to obtain a liposome solution.

[0077] (4) Preparation of the empty nanoparticle solution:

[0078] Mix the exosomes obtained in step (2) with the liposomes obtained in step (3) and PEG 8000 Mix them, and after co-incubation, purify by density gradient centrifugation to obtain a mixture. Mix this mixture with a certain concentration of Nycodenz (w / v) in PBS to produce a mixture with a slightly lower concentration. Introduce this solution, an appropriate concentration of Nycodenz PBS solution, and PBS solution into a cryogenic ultra-high-speed centrifuge for centrifugation. The purified empty nanoparticle solution is obtained from the top separation layer.

[0079] (5) Preparation of liver-targeted exosome drug-loaded nanoparticles:

[0080] Prepare a drug solution with a concentration of 20 mg / mL in advance. When preparing the drug-loaded nanoparticle solution GR-Exos@B, take the drug solution and mix it with the empty nanoparticle solution finally obtained through steps (1)-(4), perform ultrasonic treatment, and ultrafilter to remove the unencapsulated drug to obtain liver-targeted exosome drug-loaded nanoparticles;

[0081] Furthermore, in step (1), the reactants are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS);

[0082] Furthermore, in step (1), the molar ratio of the reactant EDC·HCl or NHS, bile acid derivative, and DSPE-PEG 2000 -NH2 is 1-1.5:1:1;

[0083] Furthermore, in step (2), the programs set by the high-speed centrifuge three times are 1000-10000 g, 10-40 min;

[0084] Furthermore, in step (2), when the ultra-high-speed refrigerated centrifuge separates and purifies exosomes, the set program is 180000-220,000 g, 60-120 min;

[0085] Furthermore, in step (2), when the ultra-high-speed refrigerated centrifuge precipitates and purifies the exosomes, the set program is 180000-220,000 g, 60-120 min;

[0086] Furthermore, in step (2), the different concentrations of sucrose gradients are 8%, 15%, 30%, 45%, 60% (20 mM Tris-Cl, pH 7.2);

[0087] Furthermore, in step (3), the organic solvents are chloroform-methanol mixture, trichloroethane-methanol mixture, and anhydrous ether;

[0088] Furthermore, in step (3), the emulsifiers are soybean lecithin, egg yolk lecithin, and rice bran lecithin;

[0089] Furthermore, in step (3), the pore size of the polycarbonate porous membrane is 150 - 250 nm;

[0090] Furthermore, in step (4), the setting program of the low-temperature ultra-high-speed centrifuge is 180,000 - 220,000 g, 60 - 120 min;

[0091] Furthermore, in step (5), when GR-Exos is mixed with the drug solution, the concentration ratio is 1:1 - 3;

[0092] Furthermore, in step (5), the ultrasonic program of the cell ultrasonic disruptor is 55 - 70 kHz, 15 - 30 min.

[0093] Furthermore, during application, an exosome solution and a liposome solution are prepared and mixed for incubation to prepare empty nanoparticles. A drug solution is prepared and mixed with the empty nanoparticles for incubation and then ultrasonic treatment to prepare drug-loaded nanoparticles of liver-targeting exosomes synergistically combined with liposomes. The way of using the nanoparticles is oral administration.

[0094] Previous studies have shown that nanoparticles (NPs) modified with bile acid derivatives can enter the enterohepatic circulation through endocytosis, intracellular transport, and crossing the intestine, and ultimately reach the liver to exert therapeutic effects (Chemical Engineering Journal. 2024, 485: 150129, CN115531309A). In this invention, liposomes are modified with bile acid derivatives by EDC / NHS-mediated amidation reaction, which can break through the limitations of traditional physical mixing while achieving precise targeting of the liver by using bile acid derivatives to modify liposomes and accumulating sufficiently in the liver.

[0095] This invention designs and prepares nanoparticles GR-Exos@B based on the fusion of exosomes and liver-targeting liposomes and encapsulating drugs, and breakthroughly uses high-concentration PEG 8000 in combination with Nycodenz, which can play a synergistic role, improve the fusion efficiency of exosomes and liposomes, make their fusion uniform and complete, and have low cytotoxicity, solving the problem that it is difficult to balance the fusion efficiency and product purity in traditional methods. GR-Exos@B has high-efficiency intestinal transmembrane transport ability, can effectively overcome the gastrointestinal barrier, achieve precise targeting of the liver, accumulate sufficiently, exert anti-inflammatory and antioxidant effects, synergistically improve the drug delivery efficiency, and increase the bioavailability of the encapsulated drug.

[0096] Example 1

[0097] Synthesis of DSPE-PEG 2000 -UDCA, and the synthesis process is as follows:

[0098] To synthesize DSPE-PEG incorporated with ursodeoxycholic acid (UDCA), EDC·HCl (0.432 mmol), NHS (0.432 mmol), and UDCA (0.36 mmol) were added to 6 mL of water under stirring for 30 minutes, and then DSPE-PEG 2000 -NH2 (0.36 mmol) was added, and the reaction was carried out at 37 °C for 24 hours. Then, the resulting product was transferred to a dialysis bag for one day (molecular weight cut-off (MWCO): 2.0 kDa) to remove UDCA, and then precipitated in cold diethyl ether and dried under vacuum at room temperature. The precipitated product weighed 325.3 mg, and the yield of DSPE-PEG 2000 -UDCA was approximately 86.8%. The DSPE-PEG 2000 -UDCA prepared in this example was subjected to 1H nuclear magnetic resonance analysis, and the results are as 2000 shown. Figure 1

[0099] Example 2

[0100] Extraction of ginger exosomes was carried out as follows:

[0101] The fresh ginger purchased for the experiment was fresh ginger. The extraction method was gradient centrifugation, and the instrument used was a low-temperature high-speed centrifuge. First, the fresh ginger was thoroughly washed with deionized water, chopped and stirred with PBS (NaCl, KCl, Na2HPO4, KH2PO4, weigh 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4 and dissolve in 800 mL of distilled water, adjust the solution to 7.4 with HCl, and finally add distilled water to a volume of 1 L to obtain the PBS buffer solution, and the PBS used in this article was prepared in this way) for 30 min to extract ginger juice (the ratio of ginger granules to PBS was 1 g:4 ml). The resulting ginger juice was centrifuged at 1000 g for 10 min at 4 °C, 3000 g for 20 min, and 10000 g for 40 min, and the supernatant was taken. Then, using an ultra-high-speed centrifuge, ultracentrifugation was carried out at 200,000 g for 2 h, the precipitate was resuspended with PBS, and transferred to sucrose gradients of different concentrations (the sucrose concentrations in the pH 7.2, 20 mM Tris-Cl solution were 8%, 15%, 30%, 45%, and 60% respectively). Then the mixture was centrifuged at 200,000 g for 2 h, and the bands between the 8% / 30% and 30% / 45% layers were collected respectively, washed with PBS, and centrifuged at 200,000 g for 1 h at 4 °C, and resuspended with PBS again. The final product, exosome solution G-Exos, was obtained (by controlling the amount of PBS used for resuspension, the concentration of exosome solution G-Exos was 10 mg / mL).

[0102] ​Example 3

[0103] (1) Preparation of Liposomes (RAL) and Drug-Loaded Liposomes (RAL@B)

[0104] Based on the previously reported thin-film hydration method, liver-targeted liposomes loaded with berberine hydrochloride (RAL@B) were prepared. A lipid mixture composed of soybean lecithin (10 mg / mL), DSPE-PEG-UDCA (10 mg / mL), and cholesterol (10 mg / mL) in a volume ratio of 5:1:2 was dissolved in a chloroform:methanol mixture (3:1, v / v) to form an organic solution with a liposome concentration of 10 mg / mL. Take 50 μL of this organic solution, incubate it in water at 40 °C for 1 h, evaporate it at 37 °C and 50 rpm for 30 min, and place the synthesized lipid film under nitrogen for 30 min. Dissolve the dried lipid film by adding 3 mL of anhydrous ether. Then, slowly inject the berberine hydrochloride solution (20 mg / mL, 50 μL, the 20 mg / mL berberine hydrochloride solution in this article was obtained by dissolving berberine hydrochloride in DMF) below the liquid surface using a syringe, and sonicate for 10 min to form a W / O primary solution. Evaporate the mixture at room temperature (RT) for 30 min. Hydrate the lipid film containing berberine hydrochloride above with PBS, and then rotary evaporate at room temperature for 1 h. Extrude it successively through a 200 nm polycarbonate porous membrane using a liposome extruder, and this extrusion process was repeated 20 times in total to form 10 mg / mL RAL@B with a uniform particle size.

[0105] The preparation steps of RAL are basically the same as those of RAL@B, except that water is added instead of the berberine solution to prepare 10 mg / mL of RAL.

[0106] The specific process is as follows: A lipid mixture composed of soybean lecithin (10 mg / mL), DSPE-PEG-UDCA (10 mg / mL), and cholesterol (10 mg / mL) in a volume ratio of 5:1:2 was dissolved in a chloroform:methanol mixture (3:1, v / v) to form an organic solution with a liposome concentration of 10 mg / mL. Take 50 μL of this organic solution, incubate it in water at 40 °C for 1 h, evaporate it at 37 °C and 50 rpm for 30 min, and place the synthesized lipid film under nitrogen for 30 min. Dissolve the dried lipid film by adding 3 mL of anhydrous ether. Then, dropwise add 50 μL of water and sonicate for 10 min to form a W / O primary solution. Evaporate the mixture at room temperature (RT) for 30 min. Hydrate the above lipid film with PBS, and then rotary evaporate at room temperature for 1 h. Extrude it successively through a 200 nm polycarbonate porous membrane using a liposome extruder, and this extrusion process was repeated 20 times in total to form 10 mg / mL RAL with a uniform particle size.

[0107] (2) Preparation of G-Exos@B, GR-Exos, and GR-Exos@B nanoparticles

[0108] The G-Exos with a concentration of 10 mg / mL prepared in Example 2 and the berberine hydrochloride solution with a concentration of 20 mg / mL were mixed in equal volumes and sonicated for 20 min at a power of 100 W and a frequency of 60 kHz with a 2 s on / off cycle. Subsequently, the unencapsulated berberine hydrochloride was removed by ultrafiltration using a molecular weight cut-off (MWCO) of 100 kDa to obtain G-Exos@B.

[0109] The previously prepared 10 mg / mL G-Exos, 10 mg / mL RAL, and 60% (w / v) PEG 8000 were mixed in a volume ratio of 1:1:2 and incubated at 40 °C for 2 h. Among them, PEG 8000 was dissolved in PBS to obtain 60% (w / v) PEG 8000 . The resulting mixture was purified by density gradient centrifugation according to the aforementioned method: centrifuged at 1000 g for 10 min at 4 °C, 3000 g for 20 min, 10000 g for 40 min, and the supernatant was taken; then, using an ultracentrifuge, ultracentrifuged at 200,000 g for 2 h, the precipitate was resuspended in PBS and transferred to sucrose gradients of different concentrations (the sucrose concentrations in 20 mM Tris-Cl solution with pH 7.2 were 8%, 15%, 30%, 45%, and 60% respectively). Then the mixture was centrifuged at 200,000 g for 2 h, and the bands between the 8% / 30% and 30% / 45% layers were collected separately, washed with PBS, and centrifuged at 200,000 g for 1 h at 4 °C. The precipitate was resuspended in PBS again to obtain a mixture. The mixture obtained by resuspending the precipitate in PBS was mixed with 80% Nycodenz (w / v) in a ratio of 1:1 (v / v) to produce a 40% solution (i.e., a solution containing the mixture and 40% Nycodenz (w / v)). A mixture consisting of 4 mL of the 40% solution, 2 mL of the PBS solution of 15% Nycodenz (w / v) (this solution was prepared by mixing PBS and 80% Nycodenz (w / v) in a certain ratio), and 1 mL of PBS solution was introduced into a centrifuge tube. Then, using a cryogenic ultra-high-speed centrifuge, centrifuged at a force of 200,000 g for 2 h, and the purified GR-Exos were obtained from the top separation layer. The concentration of the GR-Exos solution was adjusted to 10 mg / mL by adding PBS for dilution or concentration.

[0110] In the preparation method of GR-Exos@B in step (2) of this example, GR-Exos with a concentration of 10 mg / mL and berberine hydrochloride with a concentration of 20 mg / mL were mixed in equal volumes, and ultrasonic treatment was carried out at a power of 100 W and a frequency of 60 kHz with a 2-second on / off cycle for 20 min. Subsequently, unencapsulated berberine hydrochloride was removed by ultrafiltration with a molecular weight cut-off (MWCO) of 100 kDa to obtain GR-Exos@B.

[0111] The nanoparticles prepared in this example were subjected to particle size analysis, and the results are as Figure 2 shown.

[0112] Figure 2 It is the particle size diagram of RAL@B, RAL, G-Exos, G-Exos@B, and GR-Exos@B nanoparticles in Example 3. It can be seen from Figure 2 that the average particle size of each particle is about 200 - 300 nm.

[0113] The nanoparticles prepared in this example were subjected to scanning electron microscopy and transmission electron microscopy (TEM), and the results are shown in A - D of Figure 2 . RAL, G-Exos, GR-Exos, and GR-Exos@B are spherical, with comparable sizes, and the average values are 235.3 ± 17.1 nm, 118.4 ± 10 nm, 297.5 ± 21.4 nm, and 324.5 ± 10 nm, respectively. The drug-loaded nanoparticles are of uniform size.

[0114] Example 4

[0115] (1) Drug loading and encapsulation efficiency of nanoparticles

[0116] Mass encapsulation efficiency (EE) and drug loading (LC) are usually used to represent the drug-loading ability of nanoparticles. Among them, the drug loading is the percentage of the drug loaded in the nanoparticles and the total mass (carrier and the loaded drug), and the mass encapsulation efficiency is the mass percentage of the drug loaded in the nanoparticles and the dosed amount.

[0117] The encapsulation efficiency and drug loading of the berberine hydrochloride-loaded nanoparticles prepared in this example were measured, and the results are shown in Table 1. Compared with ginger exosomes loaded with berberine and liposomes loaded with berberine, the fusion carrier GR-Exos@B has a higher berberine loading ability (i.e., drug loading LC) and encapsulation efficiency (i.e., mass encapsulation efficiency EE).

[0118] Table 1 Drug loading, encapsulation efficiency, size, and polydispersity index of different sample amounts

[0119]

[0120] (2) Characterization of the fusion of G-Exos and GR-Exos

[0121] To evaluate the hydrodynamic diameter and ζ potential of G-Exos, RAL, G-Exos@B, RAL@B, and GR-Exos@B, dynamic light scattering analysis (DLS) was employed, and imaging was performed using transmission electron microscopy (TEM). Coomassie staining was used to analyze the protein composition of G-Exos, RAL, and GR-Exos. After determination by the BCA kit, G-Exos, RAL, and GR-Exos were prepared in loading buffer. Subsequently, the samples were heated at 95 °C for 5 minutes, and 40 μg of the samples were loaded onto an 8% SDS polyacrylamide gel. The samples were electrophoresed at 85 V for 30 minutes and 120 V for 60 minutes. Then, they were stained with Coomassie blue reagent for 1 hour, washed overnight, and subsequently observed. G-Exos were labeled with DiR (red). The labeled G-Exos and RAL were then 8000 fused (10 mg / mL G-Exos labeled with DiR, stirred for 12 h, dialyzed), and filtered through a 0.2 μm polycarbonate membrane using an extruder. The integration of G-Exos and RAL was visualized using confocal laser scanning microscopy (CLSM) and captured with a flow detector. To further explore the fusion of GR-Exos, G-Exos were labeled with Dil, and the fused GR-Exos were prepared as described above. After filtration of the nanoparticles, FRET was detected by a microplate reader at an excitation wavelength of 420 nm.

[0122] The results are as Figure 3 shown in A of Figure 3 . The average size of G-Exos measured by nanoparticle tracking analysis (NTA) was 110 nm, with a spherical morphology and uniform size, indicating the successful separation and purification of G-Exos.

[0123] (3) In vitro acid-responsive release test of drug-loaded nanoparticles

[0124] Prepare RAL@B, G-Exo@B, and GR-Exos@B according to Example 3, incubate them at pH levels of 1.2 and 6.8 for 8 hours, and measure the size of the nanoparticles using DLS. To simulate the gastrointestinal environment, introduce RAL@B, G-Exo@B, and GR-Exos@B into dialysis bags with a molecular weight cut-off (MWCO) of 12,000 Da. Then immerse these bags in 5 ml of different release media: simulated gastric fluid (SGF) containing pepsin at pH 1.2 and simulated intestinal fluid (SIF) containing trypsin at pH 6.8. Oscillate the bags continuously at 80 rpm and maintain at 37 °C. At specific time intervals, sample 200 μL of the release medium and replace it with an equal volume of fresh medium. Evaluate the concentration of berberine using HPLC. In addition, evaluate the physical stability of the nanoparticles by monitoring the changes in DLS during refrigeration (4 °C) for 7 days.

[0125] The in vitro acid-responsive cumulative release results of the drug-loaded nanoparticles are shown in Figure 4 . As Figure 4 shown in B of Figure 4 , after long-term incubation of G-Exos@B and GR-Exos@B at pH 1.2, the cumulative release amount of berberine is less than 30%, while RAL@B shows a quenched release of berberine. This indicates that the ginger exosomes prepared in the present invention can release berberine relatively continuously, effectively preventing premature drug release. Compared with G-Exos@B and RAL@B, GR-Exos@B has a slower release rate, but at pH 6.8 ( Figure 4 shown in A of

[0126] ), the cumulative release amounts of GR-Exos@B, G-Exos@B, and RAL@B within 28 h are similar, 61.2%, 73.1%, and 78.5% respectively, which further proves that the ginger exosomes prepared in the present invention have a relatively continuous release ability.

[0126] (4) Cytotoxicity experiment (MTT) of the drug-loaded nanoparticles

[0127] Prepare G-Exos@B, RAL@B, and GR-Exos@B solutions according to the methods in steps (1) and (2) of Example 3, and conduct cytotoxicity (MTT) experiments on human colon adenocarcinoma (Caco-2) and human colon adenocarcinoma (HT-29) cells. According to the different cell types and the types of nanoparticles added in the experiment, it is divided into two groups: Group A is denoted as G-Exos@B(HT-29), RAL@B(HT-29), GR-Exos@B(HT-29); Group B is denoted as G-Exos@B(Caco-2), RAL@B(Caco-2), GR-Exos@B(Caco-2). The results are shown in Figure 5 . Figure 5 A in Figure 5In [Figure 0], B is a schematic diagram of the cell survival rate in the Caco-2 cell cytotoxicity test. It can be seen that the viability of cells treated with different concentrations (50 - 500 μg / mL) of RAL@B, G-Exos@B, and GR-Exos@B remained above 80% after 24 hours of incubation. Therefore, it is demonstrated that the nanoparticles have low cytotoxicity to biological cells and are suitable for oral administration in mice without unexpected side effects.

[0128] (5) Mucus Penetration Experiment of Drug-Loaded Nanoparticles

[0129] Mix 1 mL of G-Exos, RAL, and GR-Exos (1 mg / mL) with 100 μL of the fluorescent dye DiO (1 mg / mL, dissolved in DMSO), and stir overnight at 250 rpm. During this process, DiO enters the exosome cell membrane or liposome and is thus encapsulated inside. After dialysis, G-Exos@DiO, RAL@DiO, and GR-Exos@DiO are obtained, thereby generating specific fluorescence. Culture HT-29 cells in a 12-well plate at a density of 5×10 4 cells per well and incubate until complete confluence is achieved. Subsequently, expose the cells to 100 μg / mL of G-Exos@DiO, RAL@DiO, and GR-Exos@DiO for 2 hours. After this incubation period, remove the cell culture medium and wash the cells three times with PBS. Then stain the mucus layer cells (HT-29) with 200 μL of Alexa 594-WGA diluted 1:1000 for 1 hour and wash three times with PBS. When observing, take three-dimensional images with a confocal scanning microscope and perform 3D reconstruction of the data with ZEN software.

[0130] The mucus penetration of the nanoparticles is as Figure 6 shown (it is a three-dimensional image of the cell monolayer after incubation of RAL, G-Exos, and GR-Exos with HT-29 cells). The green fluorescence is the nanoparticles, and the red fluorescence is the mucus layer. It can be seen that the green fluorescence of G-Exos and GR-Exos shows extensive distribution, and the red fluorescence of the cell layer has poor co-localization with the green fluorescence of the nanoparticles, indicating that both G-Exos and GR-Exos have penetrated through the HT-29 cell layer, showing enhanced mucus permeability, among which GR-Exos has the optimal penetration efficiency. The co-localization of the red fluorescence of the mucus layer and the green fluorescence of RAL indicates sub-optimal efficiency in penetrating mucus.

[0131] Example 5

[0132] Long-Term Administration Efficacy

[0133] (1) Long-Term In Vivo Administration of GR-Exos@B for the Treatment of NASH

[0134] Mice with high-fat diet-induced non-alcoholic hepatitis were randomly divided into five groups, with 3 mice in each group. G-Exos@B, RAL@B, and GR-Exos@B prepared in Example 3 were orally administered to the mice with high-fat diet-induced non-alcoholic hepatitis at a dose of 30 mg / kg three times a week for in vivo experiments. Among them, G-Exos@B, RAL@B, and GR-Exos@B were dissolved in water for administration, and 30 mg / kg refers to the dosage of G-Exos@B, RAL@B, or GR-Exos@B administered per kg of mice (calculated as dry weight); mice on normal diet (NM) were used as the control group. The body weights of the mice were recorded at 8 o'clock every day. After 60 days of administration, the changes in the body weights of the mice were recorded.

[0135] The process of constructing mice with high-fat diet-induced non-alcoholic hepatitis was as follows: All mice were housed in a specific pathogen-free controlled environment and provided with a standard diet for the first two weeks. Starting from the third week, their diet was supplemented with a high-fat diet, accounting for 60% of the total calories, obtained from Synergy Biotechnology Inc. The proportion of the high-fat diet in the whole rodent diet was gradually increased until the sixth week, and then the mice were fed only with the high-fat diet for another eight weeks. At the same time, a solution containing 10% D-fructose in autoclaved tap water was administered to the mice starting from the third week, and the concentration of D-fructose was gradually increased to 25% by the sixth week. As a result, the mice developed obesity and insulin resistance at the 15th week, and the development of non-alcoholic steatohepatitis (NASH) was induced after the 16th week. The body weights of the mice were about 55 - 60 g, and they showed a fasting glucose level exceeding 250 mg / dL. Measurements of the body weights and wet liver weights of the mice were performed, and at the same time, NASH-related factors (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)) in the sera of the mice were detected using ELISA kits to verify the model. This model was also used as diabetic mice.

[0136] Figure 7 Schematic diagram of the body weight levels of diabetic mice, from Figure 7 It can be seen that the administration of RAL@B, G-Exos@B, and GR-Exos@B led to a significant weight loss. The weight of the HFD group continuously increased within two months, while the final weights of all experimental groups decreased by more than 40% compared with the HFD group.

[0137] After 60 days of treatment, the changes in the secretion of inflammatory factors were studied, and the results were as Figure 8As shown, compared with the HFD, RAL@B, and G-Exos@B groups, the GR-Exos@B group showed significantly reduced levels of low-density lipoprotein (LDL) and liver function indices (ALT, ALP, AST, and TG). For example, the TG level in the RAL@B group was 1.17 mmol / L, and that in the G-Exos@B group was 1.2 mmol / L, while the GR-Exos@B group was already lower than the normal group level (1.0 mmol / L), indicating that GR-Exos@B treatment showed a reduction in inflammatory factor secretion and promoted liver function recovery.

[0138] After 60 days of treatment, the mice were dissected to obtain liver sections. The results showed that although RAL@B and G-Exos@B treatments showed a reduction in inflammatory factor secretion and promoted liver function recovery, the results of Masson staining, Oil Red O staining, and H&E staining of the liver sections indicated that hepatic steatosis was only partially recovered. In contrast, GR-Exos@B treatment showed that hepatic steatosis was almost completely normalized ( Figure 9 ).

[0139] To explore the mechanism of anti-NASH of GR-Exos@B in vivo, the levels of typical proteins involved in the oxidative stress and lipid metabolism pathways were studied. Western blot analysis performed on the livers of NASH mice treated with GR-Exos@B showed that the inhibition of markers related to lipid metabolism (SREBP-1, SCD, COA, and FAS) that promote lipid accumulation and the oxidative stress marker (CY2EP1) was significantly downregulated, ultimately improving the symptoms of NASH. In addition, the results of Western blot analysis showed that due to the HFD diet, the protein expression of p-Nrf2 / Nrf2, NOQ-1, and HO-1 was significantly reduced. The intervention of GR-Exos@B showed beneficial effects by increasing the protein levels of pNrf2 / Nrf2, NOQ-1, and HO-1 ( Figure 10 ). In addition, GR-Exos@B in the livers of NASH mice led to an enhanced ratio of p-AMPK / AMPK protein expression ( Figure 10 ). These results were consistent with the protein expression results observed at the cellular level. In addition, in the GR-Exos@B treatment group, the mRNA levels of SREBP-1 and FAS / CD95 were decreased, while the mRNA level of HO-1 showed a significant increase ( Figure 10 ).

[0140] After 60 days of long-term administration, in the detection of serum pro-inflammatory cytokine (IL-1β, IL-6, TNF-α) levels, compared with the control group, the RAL@B, G-Exos@B and GR-Exos@B treatment groups all showed a downward trend to varying degrees. Among them, the GR-Exos@B group showed the most significant inhibitory effect, and the reduction amplitude was statistically significantly different. And anti-inflammatory factors such as IL-4 and IL-10 showed the same trend. Compared with the control group, the RAL@B, G-Exos@B and GR-Exos@B treatment groups all showed an upward trend to varying degrees. Among them, the GR-Exos@B group showed the most significant promoting effect, indicating that GR-Exos@B has the ability to reduce intestinal inflammation and improve NASH symptoms( Figure 11 ).

[0141] (2) GR-Exos@B reverses the diabetic symptoms of NASH

[0142] The successfully modeled diabetic mice were randomly divided into five groups, with 3 mice in each group. G-Exos@B, RAL@B and GR-Exos@B prepared in Example 3 were orally administered to the diabetic mice three times a week at a dose of 30 mg / kg for in vivo experiments. Mice with normal diet (NM) were used as the control group. After 60 days of administration, insulin was intraperitoneally injected (i.p.) into the mice fasted after 60 days of treatment, and the blood glucose changes of the mice were continuously monitored for 120 min with a blood glucose meter. The results are as Figure 12 shown. The fasting blood glucose level showed a steady downward trend after GR-Exos@B treatment, while the fasting blood glucose values of G-Exos@B and RAL@B were similar to those of the HFD group. Treatment with GR-Exos@B effectively normalized the fasting blood glucose level, indicating the recovery of insulin resistance( Figure 12 in A). At the same time, a low insulin resistance index of the GR-Exos@B group was also shown, as Figure 12 in B. The fasting GLs of the diabetic mice in the HFD group after 60 days were 1.56 times that of the diabetic mice in the GR-Exos@B treatment group after 60 days. In addition, as Figure 12 shown in C, the blood glucose fluctuations in the treatment group gradually increased 2 hours after feeding, indicating that GR Exos@B treatment improved the fasting GLs during the extended period of NASH drug treatment and showed a significant long-term hypoglycemic effect.

[0143] The concentration of C-peptide in the bloodstream is used as a marker to evaluate the secretory activity of insulin β-cells. On the morning of the first day after the end of long-term treatment (60 days), blood was collected from the tail vein of mice, and a kit was used to detect the serum C-peptide level. Compared with the high-fat diet (HFD) group, after the intervention of G-Exos@B and RAL@B, the serum C-peptide concentration in the experimental groups increased significantly. Among them, the C-peptide level in the G-Exos@B group increased more than that in the RAL@B group, suggesting that the exosome delivery system has better efficacy in promoting C-peptide secretion. Notably, the C-peptide concentration (4.1 ng / mL) of GR-Exos@B formed by the fusion of the two delivery systems was significantly higher than that of the single delivery system group, confirming that the biological effect of the fused drug delivery system is not a simple superposition, but a synergistic effect( Figure 13 ).

[0144] After the end of long-term treatment, the mice were sacrificed, the pancreas was collected, and immunohistochemistry (IHC) analysis with insulin antibody was used to detect insulin expression. The results are as Figure 14 shown. The liver-targeting characteristics of RAL make the effect of berberine more obvious, thus further stimulating insulin secretion. The most significant increase was observed in the β-cell region close to this NM in GR-Exos@B( Figure 14 ).

[0145] To explore the in vivo mechanism of GR-Exos@B in treating diabetes, we studied the protein levels involved in the gluconeogenesis pathway. Mouse liver extracts prepared with Westing and IP buffer (i.e., the liver taken out after sacrificing the mice after treatment, and the protein running samples obtained after grinding and post-treatment) were separated on a 10% gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the purified proteins were transferred to a polyvinylidene difluoride membrane (Roche), blocked in a 5% BSA solution, and incubated with the primary antibody overnight to detect the required proteins. Then the proteins were incubated with an HRP-conjugated secondary antibody, performed with an ECL detection kit, and normalized against GAPDH or β-tubulin.

[0146] The results are as Figure 15 shown. Western blot analysis of GR-Exos@B revealed a decrease in the protein expression related to the gluconeogenesis pathway (PGC-1α and PEPCK) and an increase in the protein expression of insulin receptor (GLUT4), indicating that ginger exosomes and UDCA-modified liposomes can jointly enhance the promoting effect and enable insulin-sensitive tissues to quickly respond to changes in blood glucose( Figure 15 ).

[0147] (3) GR-Exos@B weakens hepatic macrophage infiltration and regulates macrophage polarization

[0148] Liver macrophages were transiently stained with appropriate antibodies for 30 minutes at room temperature in a light-restricted environment. Subsequently, the cells were rinsed with PBS containing 2% FBS, and then examined by flow cytometry to determine the proportion of CD86+ cells indicating M1 macrophages and the proportion of CD206+ cells indicating M2 macrophages.

[0149] The phenotypes of liver macrophages (CD86 and CD206) were evaluated by flow cytometry to study the effects of berberine, RAL, G-Exos, GR-Exos, and GR-Exos@B (the administration concentration was 100 μg / ml, incubated at 37 °C for 4 h). After treatment in each group, compared with M1 macrophages, the percentage of M2 macrophages increased. After treatment with the GR-Exos@B administration group, the expression level of the M2 macrophage marker CD206 was significantly increased compared with the control group, and the down-regulation amplitude of the M1 macrophage marker CD86 induced by the GR-Exos@B treatment group was the most significant compared with the RAL, G-Exos, and GR-Exos groups, indicating that pro-inflammatory macrophages were gradually transformed into anti-inflammatory macrophages. This transformation promoted the recovery of hepatocytes and the alleviation of inflammation ( Figure 16 ).

[0150] Add 250 μL of Westing and IP buffer to the BMDM cells cultured in a 6-well plate (the culture process was: the administration concentration of berberine, RAL, G-Exos, GR-Exos, or GR-Exos@B was 100 μg / ml, incubated at 37 °C for 4 h). Then, scrape the cells with a cell scraper at 4 °C, 10000 rpm for 10 min. Subsequently, add its supernatant to the cell lysate, incubate on a shaker at 37 °C, measure the absorbance at 562 nm, and calculate and add the respective protein supernatant buffer. Subsequently, run the cell extract on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), block, and incubate with the primary antibody overnight, and then incubate with the secondary antibody. The results are as Figure 17 shown.

[0151] Effect of GR-Exos@B on NLRP3 expression: Compared with the M1 group, the protein expression of NLRP3 in G-Exos, RAL, GR-Exos, and GR-Exos@B was significantly decreased, while the protein expression of berberine was not affected. At the same time, compared with other experimental groups, the expression level of NLRP3 in GR-Exos@B was the lowest, indicating that GR-Exos@B has the ability to alleviate inflammation by inhibiting NLRP3 signal transduction ( Figure 17 )).

[0152] (4) Long-term toxicity in vivo

[0153] Take the heart, liver, spleen, lungs, intestines, pancreas, stomach, and kidneys of the mice after 60 days of long-term administration and section them. Record the hematoxylin and eosin (H&E) staining results of the organs under a microscope. Normal mice (NM) were used as the control group.

[0154] H&E staining analysis revealed no significant tissue damage or toxic effects on the organs in the GR-Exos@B treatment group ( Figure 18 ). The orally administered drug delivery system designed in the present invention has no obvious toxic and side effects, showing its great potential as an oral drug delivery carrier.

[0155] The above embodiments are only used to illustrate the technical solutions of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form. Any technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. An exosome-fused liver-targeted liposome drug delivery system, characterized in that, The preparation process is as follows: Exosomes are fused with liver-targeting liposomes modified with bile acid derivatives to prepare empty nanoparticles, and drugs are encapsulated in the empty nanoparticles to prepare liver-targeting exosome-loaded nanoparticles, and the liver-targeting exosome-loaded nanoparticles are the exosome-fused liver-targeting liposome drug delivery system.

2. The exosome-fused liver-targeted liposome drug delivery system according to claim 1, wherein, The exosomes are derived from one of the following plants, including ginger, kaempferia galanga, yam, and pineapple.

3. The exosome-fused liver-targeting liposome drug delivery system according to claim 1, wherein The bile acid derivatives include ursodeoxycholic acid, chenodeoxycholic acid, and deoxycholic acid; the drugs are one of the drugs that can treat liver diseases, including berberine hydrochloride, morin, and semaglutide.

4. The preparation method of an exosome-fused liver-targeted liposome drug delivery system according to claim 1, characterized in that, It includes the following steps: (1) Preparation of liver-targeting ligand: Place the cholic acid derivative and the reactant in water, add DSPE-PEG 2000 -NH2, react at 35-39 °C for 20-24 h, and purify to obtain a liver-targeting ligand; (2) Extraction of exosomes: Extract ginger juice, and obtain ginger exosomes through low-temperature high-speed centrifugation and sucrose density gradient centrifugation. (3) Preparation of liver-targeting liposomes modified with bile acid derivatives: Dissolve the lipid mixture composed of an emulsifier, the liver-targeting ligand obtained in step (1), and cholesterol in an organic reagent, and prepare liver-targeting liposomes modified with bile acid derivatives by extrusion. (4) Preparation of empty nanoparticle solution: Mix the exosomes obtained in step (2) with the liver-targeting liposomes modified with bile acid derivatives obtained in step (3) and PEG 8000 and incubate them together. After purification by density gradient centrifugation, a mixture is obtained, where the PEG 8000 concentration is 60 ± 3% w / v; mix this mixture with Nycodenz at 75 - 85% w / v to form a Nycodenz solution with a first concentration containing the mixture. The Nycodenz solution with the first concentration, the Nycodenz solution with the second concentration, and PBS form a gradient solution. After ultracentrifugation, the top separation layer is separated to obtain purified empty nanoparticles; the Nycodenz concentration gradient decreases in the range of 80% to 15%; the first concentration is lower than 75 - 85% w / v; the second concentration is lower than the first concentration; (5) Preparation of ligand-modified drug-loaded nanoparticles: Take an equal volume of the drug solution and the empty nanoparticle solution and mix them to obtain liver-targeting exosome-loaded nanoparticles; the concentration ratio of the empty nanoparticle solution to the drug solution is 1:1 - 3.

5. The preparation method of an exosome-fused liver-targeted liposome drug delivery system according to claim 4, characterized in that, In step (1), the reactants are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS); the molar ratio of the reactants EDC·HCl or NHS, the cholic acid derivative, and DSPE-PEG 2000 -NH2 is 1 - 1.5:1:1 - 1.

5.

6. The preparation method of an exosome-fused liver-targeted liposome drug delivery system according to claim 4, wherein Step (2) is specifically as follows: Extract ginger juice, centrifuge at 800 - 1200 g for 5 - 10 min at 0 - 4 °C, centrifuge at 2500 - 3500 g for 15 - 20 min, centrifuge at 9000 - 11000 g for 20 - 40 min; ultracentrifuge at 180,000 - 200,000 g for 1 - 2 h, resuspend, and transfer to sucrose solutions with different gradient concentrations for centrifugation. The gradient concentrations of sucrose are 8%, 15%, 30%, 45%, and 60% respectively. Then centrifuge, collect the bands between the 8% / 30% layer and the 30% / 45% layer, wash, centrifuge, and resuspend to obtain ginger exosomes.

7. The preparation method of an exosome-fused liver-targeted liposome drug delivery system according to claim 4, wherein In step (3), the emulsifier, liver-targeting ligand, and cholesterol are mixed in a mass ratio of 1 - 5:1 - 2:

2.

8. The preparation method of an exosome-fused liver-targeted liposome drug delivery system according to claim 4, characterized in that, In step (4), the volume ratio of exosomes, liver-targeted liposomes modified with bile acid derivatives, and PEG 8000 is 1-2:1:1-2.

9. The preparation method of an exosome-fused liver-targeted liposome drug delivery system according to claim 4, wherein In step (5), the drug is encapsulated in the empty nanoparticles by ultrasound, and the ultrasound parameters are 55 - 70 kHz for 15 - 30 min.

10. Use of the exosome-fused liver-targeting liposome drug delivery system according to any one of claims 1 - 3 or the preparation method according to any one of claims 4 - 9 in the preparation of drugs for treating non-alcoholic fatty hepatitis and type II diabetes.

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